Research Article |
Corresponding author: Ruttapon Srisonchai ( ruttasr@kku.ac.th ) Corresponding author: Thomas Wesener ( t.wesener@leibniz-lib.de ) Academic editor: Nesrine Akkari
© 2024 Ruttapon Srisonchai, Natdanai Likhitrakarn, Chirasak Sutcharit, Thomas Wesener.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Srisonchai R, Likhitrakarn N, Sutcharit C, Wesener T (2024) Integrative taxonomy reveals two new giant pill-millipedes of the genus Zephronia Gray, 1832 from eastern Thailand (Diplopoda, Sphaerotheriida, Zephroniidae). ZooKeys 1212: 29-64. https://doi.org/10.3897/zookeys.1212.126536
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A large amount of material of the millipede genus Zephronia Gray, 1832 was collected during 2014–2023 from many parts of eastern Thailand. An integrative study of morphological characters and genetic data (COI gene) revealed two new species: Z. chantaburiensis Srisonchai & Wesener, sp. nov. and Z. macula Srisonchai & Wesener, sp. nov. The two new species clearly differ from other congeners by their unique characteristics, especially in their colour pattern and telopod shape. The interspecific genetic distances of the 658 bp COI gene barcoding fragment between these new species and all other species of giant pill-millipede from Thailand, Laos and Cambodia are 12.01–23.49% for Z. chantaburiensis sp. nov. and 17.93–25.13% for Z. macula sp. nov. While relationships among species remain preliminary, the phylogenetic tree shows that species of Zephronia are interspersed with species of Sphaerobelum Verhoeff, 1924 and Prionobelum Verhoeff, 1924. Phylogenetic analyses place both new species in a clade termed Zephronia s.s., which receives support also from morphological data, showing a unique position of the organ of Tömösváry. Z. macula sp. nov. appears to occur over a broad distribution whereas Z. chantaburiensis sp. nov. was found only at the type locality. Given that all known records are in the eastern part of Thailand, we thus regard both species as endemic. Morphological illustrations based on SEM micrographs and a distribution map are also provided.
Biodiversity, limestone, Myriapoda, Southeast Asia, taxonomy
Intensive research on the millipedes (class Diplopoda) in Thailand began in 2007 after the discovery of a beautifully ornamented and endemic creature, the shocking pink dragon millipede Desmoxytes purpurosea Enghoff, Sutcharit & Panha, 2007 which has been endorsed by the public and has inspired further studies on several millipede groups (
Currently, three genera of giant pill-millipedes (order Sphaerotheriida) are known from Thailand: Sphaerobelum Verhoeff, 1924 with four species (see
The genetic approach based on DNA barcoding has been widely used for species delimitation in millipedes in recent years, but it is considered more reliable when used in combination with morphological evidence. Previous phylogenetic studies have notably provided insights into species discrimination for Zephronia (
Considering the newly collected material, two morphologically distinct groups of specimens were observed during fieldwork. Given their visible morphological differences from the other known Thai giant pill-millipede species, the suspicion arose that they may represent undescribed species. In this paper, we integratively describe two new species from the eastern part of Thailand based on morphological traits using scanning electron microscopy and phylogenetic analyses based on mitochondrial COI sequences, in order to confirm their status and compare them with the known congeneric species from Thailand, Laos and Cambodia.
The material was obtained from the collections of the Chulalongkorn University Museum of Zoology (
The map present herein was generated based on the background photo from the Elastic Terrain Map (
The format of the morphological descriptions follows
The holotypes, as well as the paratypes, are deposited in the Chulalongkorn University Museum of Zoology, Thailand (
List of abbreviations used in the description and figures:
3it 3-combed inner tooth of mandible,
bu bursa of vulva,
co condylus of mandible,
cp cuticular impression of endotergum;
Cp central pad of gnathochilarium,
cr-t crenulated teeth on telopods,
ct central tooth,
cx coxa,
et external tooth of epipharynx,
Et external tooth of mandibular gnathal lobe,
fe femur,
ia inner area of endotergum;
Ia inner area of mandible,
il incisura lateralis of the head,
imf immovable finger of telopod;
ip inner palpi of gnathochilarium,
ll lamellae lingulales of gnathochilarium,
ma middle area of endotergum;
me mentum of gnathochilarium,
ml membranous lobe,
mp molar plate of mandible,
op operculum of vulva,
pl pectinate lamellae of mandible,
pm posterior margin of endotergum;
po postfemur,
pre prefemur,
rsp row of spines,
sc sensory cone,
scl-s sclerotized spots,
st stipites of gnathochilarium,
st-pl stigmatic plate,
ta tarsus,
ti tibia.
Specimen repositories and others
NHMD Natural History Museum of Denmark, Denmark;
Body parts of specimens for scanning electron microscopy (SEM) were carefully dissected under a stereomicroscope, placed in a dry cabinet for 24 hours, mounted on aluminium stubs, and then coated with gold. Objects were examined under a high vacuum in JEOL, JSM-5410 LV at the faculty of Science, Khon Kaen University. All figures were assembled and adjusted in Adobe Photoshop CS6.
DNA of 11 specimens (9 specimens of the two new species and 2 specimens of Z. siamensis) were extracted from the legs using the NucleoSpin Tissue Kit. We analysed the mitochondrial cytochrome c oxidase subunit I (COI) as a DNA barcoding gene. The polymerase chain reactions were carried out using LCO-1490 as forward and HCO-2198 as reverse primers — LCO-1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO-2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (
We analysed a total of 84 sequences comprising 11 sequences generated from this study and 73 sequences obtained from GenBank, including all known giant pill-millipede species of Zephronia from Thailand, Cambodia, and Laos. Sphaerobelum spp., Prionobelum spp., Cryxus ovalis (Linnaeus, 1758), Epicyliosoma sp., Arthrosphaera brandti (Humbert, 1865), Sphaeromimus splendidus Wesener & Sierwald, 2005 and Glomeris marginata (Villers, 1789) were used as outgroups. All new sequences were submitted to GenBank with accession numbers provided in Table
Lists of Zephronia, Sphaerobelum, Prionobelum and Cryxus species analysed in this study and their COI accession numbers.
Species | COI accession number | Voucher code | Locality | Reference | |
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Ingroups | |||||
1 | Z. chantaburiensis sp. nov. | PP754582 |
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Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim | This study |
2 | Z. chantaburiensis sp. nov. | PP754583 |
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Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim | This study |
3 | Z. chrysomallos Bhansali & Wesener, 2022 | OM509649 |
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Thailand, Kanchanaburi Province, Sai Yok District, Sai Yok Noi Waterfall |
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4 | Z. dawydoffi Attems, 1953 | MK330971 |
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N/A |
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5 | Z. erawani Bhansali & Wesener, 2022 | OM509650 | NHMD K56x9 | Thailand, Kanchanaburi Province, Si Sawat District, 50 km W of Kanchanaburi, Erawan Waterfall |
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6 | Z. golovatchi Srisonchai, Sutcharit & Likhitrakarn, 2021 | OM509646 |
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Thailand, Nakhon Ratchasima Province, Pak Chong District |
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7 | Z. golovatchi Srisonchai, Sutcharit & Likhitrakarn, 2021 | OM509647 |
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Thailand, Nakhon Nayok Province, Khao Yai National Park |
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8 | Z. hui Liu & Wesener, 2022 | OP339790 | SCAU YGM03 | China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Yamugou Scenic Area |
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9 | Z. hui Liu & Wesener, 2022 | OP339791 | SCAU YGM02 | China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Yamugou Scenic Area |
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10 | Z. lannaensis Likhitrakarn & Golovatch, 2021 | OM509629 |
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Thailand, Chiangmai Province, Mueang District, Doi Suthep |
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11 | Z. lannaensis Likhitrakarn & Golovatch, 2021 | OM509630 |
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Thailand, Chiangmai Province, Mae Rim District, Traidhos School Campus |
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12 | Z. lannaensis Likhitrakarn & Golovatch, 2021 | OM509631 |
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Thailand, Chiangmai Province, Mae Rim District, Mae Sa Valley |
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13 | Z. lannaensis Likhitrakarn & Golovatch, 2021 | OM509632 | NHMD K57B | Thailand, Chiangmai Province, Mueang District, Doi Suthep, Me Sa Waterfall |
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14 | Z. lannaensis Likhitrakarn & Golovatch, 2021 | OM509633 | MHNG 3B | Thailand, Chiangmai Province, Mueang District, Doi Suthep |
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15 | Z. laotica Wesener, 2019 | MK330977 |
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Laos, Champasak Province, east of Mekong, Garden of Erawan Riverside Hotel |
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16 | Z. macula sp. nov. | PP754589 |
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Thailand, Sra Kaeo Province, Mueang Sra Kaeo District, Wat Tham Khao Maka | This study |
17 | Z. macula sp. nov. | PP754590 |
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Thailand, Sra Kaeo Province, Mueang Sra Kaeo District, Wat Tham Khao Maka | This study |
18 | Z. macula sp. nov. | PP754584 |
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Thailand, Chantaburi Province, Khlung District, Thaeo Khlong Khlung Monastery | This study |
19 | Z. macula sp. nov. | PP754585 |
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Thailand, Chantaburi Province, Khlung District, Thaeo Khlong Khlung Monastery | This study |
20 | Z. macula sp. nov. | PP754586 |
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Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim | This study |
21 | Z. macula sp. nov. | PP754587 |
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Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim | This study |
22 | Z. macula sp. nov. | PP754588 |
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Thailand, Chantaburi Province, Tha Mai District, Wat Khao Sukim | This study |
23 | Z. medongensis Zhao & Liu, 2022 | OP339793 | SCAU XZ01 | China, Xizang Autonomous Region, Medog County |
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24 | Z. ovalis Gray, 1832 | JX486068 |
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Vietnam, Dong Nai Province, Cat Tien National Park |
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25 | Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 | OM509643 |
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Thailand, Ratchaburi Province, Ratchaburi-Photharam Districts |
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26 | Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 | OM509644 | MHNG 3A | Thailand, Ratchaburi Province, Chom Bueang District, Tham Kao Bin Forest Park |
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27 | Z. panhai Srisonchai, Sutcharit & Likhitrakarn, 2021 | OM509645 |
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Thailand, Ratchaburi Province, Ratchaburi-Photharam Districts |
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28 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509634 |
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Thailand, Chiang Mai Province, Chiang Dao District, Padeng Lodge |
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29 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509635 | MYR3500 | Thailand, Chiangmai Province, Mueang District, Doi Suthep |
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30 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509636 | SMF | Thailand, Chiang Mai Province, Chiang Dao District, Tham Houay Luk |
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31 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509637 | SMF | Thailand, Chiang Mai Province, Chiang Dao District, Doi Chiang Dao, Ma Lee’s Resort |
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32 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509638 | SMF | Thailand, Chiang Mai Province, Chai Prakan District, Tham Ngam |
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33 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509639 |
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Thailand, Chiang Mai Province, Chiang Dao |
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34 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509640 | MHNG 5G | Thailand, Lamphun Province, Mae Tha District, Doi Khuntan National Park |
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35 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509641 | MHNG 5I | Thailand, Chiang Mai Province, Chiang Dao District, Doi Chiang Dao |
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36 | Z. phrain Likhitrakarn & Golovatch, 2021 | OM509642 | NHMD K35 | Thailand, Chiang Mai Province, Ban Musue |
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37 | Z. siamensis Hirst, 1907 | JX486067.2 | FMNH-INS-72669 | Thailand, Chonburi Province, Sichang District, Koh Sichang |
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38 | Z. siamensis Hirst, 1907 | OR530089 |
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Thailand, Chonburi Province, Sichang District, Koh Sichang |
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39 | Z. siamensis Hirst, 1907 | PP754592 |
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Thailand, Chachoengsao Province, Phanom Sarakham District, Wat Khao Hin Sorn | This study |
40 | Z. siamensis Hirst, 1907 | PP754591 |
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Thailand, Srakaeo Province, Khao Chakan District, Wat Tham Khao Chan | This study |
41 | Zephronia sp. (K45 Aow Noi Temple) | MW898741 | NHMD K55 | Thailand, Prachuap Kiri Khan Province, Mueang District, Aow Noi Temple |
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42 | Zephronia sp. (Aow Noi Temple) | MW898742 |
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Thailand, Prachuap Kiri Khan Province, Mueang District, Aow Noi Temple |
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43 | Z. viridisoma Rosenmejer & Wesener, 2021 | MW898739 | NHMD 621695 | Thailand, Nakhon Si Thammarat Province, Sichon District, Khao Lark Waterfall |
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44 | Z. viridisoma Rosenmejer & Wesener, 2021 | MW898740 |
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Thailand, Nakhon Si Thammarat Province, Sichon District, Khao Lark Waterfall |
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45 | Z. zhouae Zhao & Liu, 2022 | OP339794 | SCAU YN02 | China, Yunnan Province, Diqing Tibetan Autonomous Prefecture, Weixi County, Laowo Village |
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Outgroups | |||||
46 | S. aesculus Rosenmejer & Wesener, 2021 | MW898737 | NHMD 621693 | Thailand, Phuket Province, Kathu District, Forest |
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47 | S. cf. aesculus Rosenmejer & Wesener, 2021 | MW898738 | NHMD 621694 | Thailand, Nakhon Si Thammarat Province, Khao Luang NP |
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48 | S. benquii Liu & Wesener, 2022 | OP339792 | SCAU MMY01 | China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Guanhe Village, Maomaoyan |
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49 | S. bolavensis Wesener, 2019 | MK330982 | MHNG LT-10/24 | Laos, Champasak Province, Bolaven Plateau, 3 km S of Ban Nong Luang, Tad Kameud |
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50 | S. denticulatum Wesener, 2019 | MK330984 | MHNG LT-10/12 | Laos, Oudomxai Province, ca 3 km E of Tad Lak 11, SE of Oudomxai city |
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51 | S. huzhengkuni Zhao, Yu & Liu, 2020 | MT657327 | SCAU SP02 | China, Guizhou Province, Tongren City, Fanjingshan National Nature Reserve |
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52 | S. huzhengkuni Zhao, Yu & Liu, 2020 | MT657328 | SCAU SP03 | China, Guizhou Province, Tongren City, Fanjingshan National Nature Reserve |
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53 | S. lachneeis Wesener, 2019 | MK330983 | MHNG LT-10/12 | Laos, Oudomxai Province, ca 3 km E of Tad Lak 11, SE of Oudomxai city |
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54 | S. laoticum Wesener, 2019 | MK330975 | SMF | Laos, Vientiane Province, Vang Vieng |
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55 | S. meridionalis Bhansali & Wesener, 2022 | OM509648 | MHNG 4B-2 | Thailand, Yala Province, Bannang Sata District, Bang Lang National Park, near Than To Waterfall |
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56 | S. nigrum Wesener, 2019 | MK330976 | SMF | Laos, Champasak Province, Muang Bachieng, Ban Lak 35, Tad Etu |
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57 | S. peterjaegeri Wesener, 2019 | MK330972 | SMF SD553 | Laos, Luang Prabang Province, SE Luang Prabang, Nam Khan, Ban Pak Bak, Houay Kho |
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58 | S. phouloei Wesener, 2019 | MK330974 | ZMUC00040257 | Laos, Houaphan Province, Phou Loei |
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59 | S. schwendingeri Wesener, 2019 | MK330978 | MHNG LT-10/03 | Laos, Vientiane Province, trail to Tham Pou Kham, W. of Vang Vieng |
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60 | S. schwendingeri Wesener, 2019 | MK330981 | SMF | Laos, Vientiane Province, Vang Vieng, Tham Pou Kham |
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61 | Sphaerobelum sp. L07 | MK330979 | ZMUC00040261 | Laos, Khammouane Province, Ban Khounkham [Khun Kham] (Nahin) |
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62 | Sphaerobelum sp. L10 | MK330980 | SMF | Laos, Vientiane Province, Vang Vieng, W. of Nam Song, Tham Nam Or Khem |
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63 | S. spinatum Wesener, 2019 | MK330973 | ZMUC00040258 | Laos, Vientiane Province, Phou Khao Khouay |
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64 | S. truncatum Wongthamwanich, 2012 | JN885184 | FMNH-INS 0000 072 674 | Thailand, Nan Province, Song Khwae District, Na Rai Luang Subdistrict, Pang Hi Village |
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65 | S. turcosa Srisonchai & Pimvichai, 2023 | OR530087 |
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Thailand, Loei Province, Mueang Loei District, Phu Pha Lom Forest Park |
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66 | S. turcosa Srisonchai & Pimvichai, 2023 | OR530087 |
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Thailand, Loei Province, Mueang Loei District, Phu Pha Lom Forest Park |
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67 | S. tujiaphilum Zhao & Liu, 2022 | OP339783 | SCAU SD02 | China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village |
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68 | S. tujiaphilum Zhao & Liu, 2022 | OP339784 | SCAU SD01 | China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village |
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69 | S. tujiaphilum Zhao & Liu, 2022 | OP339785 | SCAU BHS01 | China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Baiheshan Village |
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70 | S. tujiaphilum Zhao & Liu, 2022 | OP339786 | SCAU JXT01 | China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Jiang-xitun Village |
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71 | S. tujiaphilum Zhao & Liu, 2022 | OP339787 | SCAU JXT02 | China, Guizhou, Tongren City, Jiangkou County, Taiping Town, Jiang-xitun Village |
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72 | S. tujiaphilum Zhao & Liu, 2022 | OP339788 | SCAU SD03 | China, Guizhou, Tongren City, Jiangkou County, Guanhe Town, Sidu Village |
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73 | S. tujiaphilum Zhao & Liu, 2022 | OP339789 | SCAU DW01 | China, Guizhou, Tongren City, Jiangkou County, Dewang Town, Xiaobang Village |
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74 | Arthrosphaera brandtii (Humbert, 1865) | FJ409915 | FMNH-INS 8650 | Tanzania, Usambara hills |
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75 | Cryxus ovalis (Linnaeus, 1758) | JX486069.2 |
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Vietnam, Dong Nai Province, Cat Tien National Park |
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76 | Epicyliosoma sp. | AF218270 | NA | NA |
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77 | Glomeris marginata (Villers, 1789) | FJ409909 |
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Germany, Bonn, Venusberg |
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78 | Prionobelum inthanonense Donworth & Wesener, 2024 | PP297645 | MHNG 4E-2 | Thailand, Chiang Mai Province, Chom Thong District, Doi Inthanon National Park |
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79 | Prionobelum inthanonense Donworth & Wesener, 2024 | PP297646 | MHNG 7A | Thailand, Chiang Mai Province, Chom Thong District, Doi Inthanon National Park |
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80 | Prionobelum naevium Donworth & Wesener, 2024 | PP297647 | MHNG 4B-1 | Thailand, Yala Province, Than To District, Bang Lang National Park, Than To Waterfall |
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81 | Prionobelum naevium Donworth & Wesener, 2024 | PP297648 | NHMD 1184671 | Thailand, Yala Province, Than To District, Bang Lang National Park |
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82 | Prionobelum naevium Donworth & Wesener, 2024 | PP297649 | NHMD 1184672 | Thailand, Yala Province, Than To District, Bang Lang National Park |
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83 | Prionobelum naevium Donworth & Wesener, 2024 | PP297650 | NHMD 1184673 | Thailand, Yala Province, Than To District, Bang Lang National Park |
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84 | Sphaeromimus splendidus Wesener & Sierwald, 2005 | FJ409917 | FMMC-INS 6702 | Madagascar, Sainte Luce S9 |
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The inspection of sequencing chromatograms was performed in MEGA 7 (
The phylogenetic trees were generated under two approaches: maximum likelihood (ML) and Bayesian inference (BI). For ML, the analysis was conducted using IQ-tree on XSEDE 1.6.6 (
The final aligned COI sequences were composed of 658 base pairs, and the sequence annotation contained 375 variable sites, 263 conservative sites and 312 sites were parsimony informative. The percentual distances between outgroups (Arthrosphaera brandtii + Epicyliosoma sp. + Arthrosphaera brandti + Sphaeromimus splendidus) and Zephronia + Sphaerobelum + Prionobelum + Cryxus were 25.11–46.39%. The intergeneric distances between Zephronia and Sphaerobelum ranged between 7.19 to 31.62%, between 19.13 to 28.63% for Zephronia and Prionobelum, and between 21.19 to 28.75% for Zephronia and Cryxus. The interspecific distances within Zephronia, Sphaerobelum, Prionobelum each ranged from 9.91–29.93%, 10.86–31.21% and 20.60–22.12%, respectively. The intraspecific distances of Z. chantaburiensis sp. nov. and Z. macula sp. nov. ranged between 0–0.15% and 0.15–4.40%, respectively. The two new species were separated from each other by an interspecific p-distance of 18.94–19.71%. Information regarding genetic distances is presented in the Suppl. material
BI and ML methods returned different topologies, especially at deep nodes (Fig.
Phylogenetic tree based on Bayesian Inferences (BI). Numbers at nodes indicate Bayesian posterior probabilities (PP)/ bootstrap support (BS). The dash “-” at the nodes refer to a different topology in the BI analysis compared to ML (see Supplemental Material 2). Colours denote the two new species (Zephronia chantaburiensis sp. nov. and Zephronia macula sp. nov.), corresponding to the live photograph. Scale bar represents substitutions/site.
The low support values of deep nodes in our trees based on the COI gene alone do not allow for a conclusion on the relationships among the species of Zephronia as well as of those in Sphaerobelum, Prionobelum, and Cryxus. The preliminary data, however, questions the monophyly of these genera.
According to the results, the trees from both BI and ML clearly show that the genus Zephronia is recovered as polyphyletic, with the clades formed by Z. viridisoma and Zephronia sp. (Aow Noi Temple) as a well-supported sister group to all other species of Zephronia and Sphaerobelum located in a trichotomy with a branch supporting Prionobelum and Cryxus Leach, 1814 (PP = 1.0, BS = 98). Z. hui and Z. phrain cluster among species of Sphaerobelum, but with low statistical support. Morphologically aberrant species of Sphaerobelum cluster with Prionobelum inthanonense. All other Thai, Laotian and Cambodian Zephronia s.s. are in a well-supported monophylum in both BI and ML (PP = 0.97, BS = 82) including the two new species described here (Fig.
Family Zephroniidae Gray, 1843
Subfamily Zephroniinae Gray, 1843
Tribe Zephroniini Gray, 1843
Differs from all other genera of Zephroniinae by the combination of the following characters: 1) Body length 18–50 mm. 2) Antennae flattened laterally, usually axe-shaped, with numerous (> 4) apical cones. 3) Endotergum (tergite underside) weakly modified: posterior margin (pm) usually flat; outer area (os) without setae; marginal bristles arranged in 1–5 rows; middle area (ma) often with a single row of circular cuticular impressions. 4) Tarsi of legs 5–21 often with more than one apical spine (with very few species with only 1) and several ventral spines. 5) Anterior telopods with four podomeres distal to syncoxite; telopoditomere 2 with a large, curved process forming a clamp-like; telopoditomeres 3 and 4 simple. 6) Posterior telopods with four telopoditomeres; immovable finger (process of telopoditomere 2 slender, apically curved; telopoditomeres 3 and 4 with two membranous lobes; telopoditomere 3 also with a row of conspicuous, crenulated teeth, larger than elopoditomere 4; telopoditomere 4 with few (one or two) sclerotized spines. 7) Female vulvae conspicuous, slender; operculum usually round.
Zephronia is one of the most species-rich genera of the family with more than 50 species currently placed in the genus. Numerous species currently placed in the genus are in need of a revision, some only known from the female, and might be placed in separate genera in the future. The posterior telopod in Zephronia consists of four podomeres, unlike Castanotherium Pocock, 1895 whose species have three podomeres only. Podomere 4 of the posterior telopod in Zephronia is not strongly curved or overlapping the process of podomere 2 as in Cryxus Leach, 1814. The process of podomere 2 in Zephronia is never apically enlarged or swollen like in Sphaerobelum Verhoeff, 1924. The osterior telopod on podomere 3 in Zephronia has crenulated teeth, unlike Kophosphaera Attems, 1935 which lacks sclerotized teeth or spines entirely. The posterior telopod of Zephronia species is identical to those of species of Sphaeropoeus Brandt, 1833, Prionobelum Verhoeff, 1924, Tigridosphaera Jeekel, 2000, and Indosphaera Attems, 1935. Zephronia species differ from species of Sphaeropoeus and Prionobelum in the anterior telopods, lacking the characteristic processes on joints three (Sphaeropoeus) and four (Prionobelum). Zephronia species differ from Indosphaera in the coxae of leg 2 in females being separate, not fused. Zephronia species are currently impossible to distinguish from species of Tigridosphaera as that species-poor genus is in need of revision. Females of Zephronia differ in their shape of the operculum of the vulva from species of Indosphaera, where it is much lower than in Zephronia species, as well as from species of Sphaeropoeus and Prionobelum where the operculum is greatly enlarged and almost rectangular in shape.
Differing from all Zephronia s.l. by the position of the organ of Tömösváry, which is located at the brim and not inside the antennal groove as in all other known Sphaerotheriida. Zephronia s.s. includes the type species of the genus, Z. ovalis, as well as Z. chantaburiensis sp. nov., Z. chrysomallos, Z. dawydoffi, Z. erawani, Z. enghoffi, Z. golovatchi, Z. hui Liu & Wesener, 2022, Z. konkakinhensis Semenyuk, Golovatch & Wesener, 2018, Z. lannaensis, Z. laotica, Z. macula sp. nov., Z. medongensis Zhao & Liu, 2022, Z. montis Semenyuk, Golovatch & Wesener, 2018, Z. panhai, Z. siamensis and Z. zhouae Zhao & Liu, 2022. See more details for the genus in
Holotype
• ♂ (
• 26 juveniles (
The position of the organ of Tömösváry at the brim and not inside the antennal groove (Fig.
Zephronia chantaburiensis sp. nov., ♂ paratype (
Measurements : Male holotype. Body length 20 mm. Width, of thoracic shield 9 mm, of tergite 7 = 10 mm (= broadest). Height of tergite 7 = 7 mm (= highest). Males: body length = 19–23 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–10 mm. Height of tergite 7 = 6–8 mm. Females: body length = 19–22 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–11 mm. Height of tergite 7 = 6–8 mm (= highest).
Colouration
(Fig.
Head
(Fig.
Antennae
(Figs
Epipharynx
(Fig.
Gnathochilarium
(Fig.
Mandibles (gnathal lobe) (Fig.
Tegument
(Figs
Zephronia chantaburiensis sp. nov., ♂ paratype (
Collum
(Figs
Thoracic shield
(Fig.
Midbody tergite
(Fig.
Anal shield
(Fig.
Endotergum of thoracic shield and midbody tergite
(Fig.
Pleurite (laterotergite)
(Fig.
Zephronia chantaburiensis sp. nov. SEM. A–G, L ♂ paratype (
Subanal plate of female
(Fig.
Stigmatic plates
(Fig.
Legs
(Fig.
Male sexual characters
(Fig.
Anterior telopods
(Fig.
Posterior telopods
(Figs
Zephronia chantaburiensis sp. nov., ♂ paratype (
Female sexual characters
(Fig.
Negligible colour variation is observed in the anal shield between different living specimens. Generally, they are dark brown in most specimens and pale brown in others. Females are typically of the same size as males.
Zephronia chantaburiensis sp. nov. is currently known only from the type locality in Chantaburi Province and is therefore here regarded as an endemic species. The new species was encountered during the day time in evergreen forest in granitic habitat (Fig.
The name is an adjective referring to the province (Chantaburi) where the type locality is located.
Holotype
• ♂ (
• 29 juveniles (
The position of the organ of Tömösváry in this small Zephronia with an axe-shaped antennomere 6 identifies Z. macula sp. nov. as a member of the Zephronia s.s. species-group (
Measurements : Male holotype. Body length 18 mm. Width, of thoracic shield 8 mm, of tergite 7 = 9 mm (= broadest). Height of tergite 7 = 6 mm (= highest). Males: body length = 18–20 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 8–10 mm. Height of tergite 7 = 6–7 mm. Females: body length = 18–20 mm. Width, of thoracic shield = 8–9 mm, of tergite 7 = 9–10 mm. Height of tergite 7 = 6–8 mm (= highest).
Colouration
(Fig.
Head
(Fig.
Zephronia macula sp. nov., ♂ paratype (
Antennae
(Figs
Epipharynx
(Fig.
Gnathochilarium
(Fig.
Mandibles (gnathal lobe) (Fig.
Tegument
(Figs
Zephronia macula sp. nov., ♂ paratype (
Collum
(Figs
Zephronia macula sp. nov. — SEM A–G, L ♂ paratype (
Thoracic shield
(Fig.
Midbody tergite
(Fig.
Anal shield
(Fig.
Endotergum of thoracic shield and midbody tergite
(Fig.
Pleurite (laterotergite)
(Fig.
Subanal plate of female
(Fig.
Stigmatic plates
(Fig.
Legs
(Fig.
Male sexual characters
(Fig.
Anterior telopods
(Fig.
Zephronia macula sp. nov., ♂ paratype (
Posterior telopods
(Figs
Endoterga of thoracic shields and midbody tergites — SEM A–D Zephronia chantaburiensis sp. nov. (♂ paratype,
Distribution and localities of Zephronia species in Thailand. Number inside the circle indicates species: 1 = Z. chantaburiensis sp. nov.; 2 = Z. chrysomallos; 3 = Z. enghoffi; 4 = Z. erawani; 5 = Z. golovatchi; 6 = Z. lannaensis; 7 = Z. macula sp. nov.; 8 = Z. panhai; 9 = Z. phrain; 10 = Z. siamensis; 11 = Z. viridisoma.
Female sexual characters
(Fig.
Intrapopulational variation was found in which most specimens have a brown/reddish brown (majority) colour, while the others are greenish brown (minority). In addition, the posterior margin of subanal scale is in most specimens wide, whereas some specimens possess a quite narrow margin.
This species is widely distributed across eastern Thailand (>150 km). Most specimens were collected from locations with granitic rock habitats, while a few localities were in forested limestone habitats (Fig.
The name is a noun referring to the pattern of spots on the body.
Two new species of giant pill-millipedes from Thailand have been integratively described on the basis of morphological characters and genetic information. These new species are taxonomically assigned to the genus Zephronia due to the combination of the distinct characters of the antennae, endotergum, tarsi of legs 5–21, anterior telopods, posterior telopods and female vulvae; they share a few morphological traits with most of their congeners (viz., body size ca 20 mm with brown colour, one row of bristles on endotergum). They clearly belong to the Zephronia s.s. species group (Z. chrysomallos, Z. dawydoffi, Z. erawani, Z. enghoffi, Z. golovatchi, Z. hui, Z. konkakinhensis, Z. lannaensis, Z. laotica, Z. medongensis, Z. montis, Z. ovalis, Z. panhai, Z. siamensis and Z. zhouae), both morphologically and genetically (Fig.
The interspecific distances based on the 658 bp COI barcoding fragment of the two new species compared to its congeners are quite large, with 18.94–26.82% in Z. chantaburiensis sp. nov. and 17.93–25.13% in Z. macula sp. nov. While the usual range of interspecific genetic distances for species discrimination in most giant pill-millipedes is from 8 to 21%, the observed distances in this study are higher than between most previously recognized species within the genus. Our analysis of the interspecific distances is consistent with those reported in recently described Zephronia species (
We conducted four intensive surveys throughout the eastern part of Thailand from 2019 to 2023. According to the distribution record, Z. macula sp. nov. has a wide range, covering the majority of eastern Thailand (Fig.
The present work adds two endemic species of Zephronia, resulting in a total of 11 species for the genus in Thailand (51 valid species worldwide). This discovery has also greatly expanded the known range of the genus in the far eastern part of Thailand, but leaves a gap of more than 400 km without any record of Zephronia along the coast of Thailand in the closest country area (Cambodia) where the Cardamom Mountain Range lies. Searching in still unexplored places in Thailand and neighboring counties would be fruitful in revealing the hidden diversity of the genus and the evolution of the taxon. It is believed that as investigations and intensive analyses go further, more new species will certainly be discovered.
The curators and staffs of
The authors have declared that no competing interests exist.
No ethical statement was reported.
This work (Grant No. RGNS65-056) was supported by the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI), and partly granted by the National Science, Research and Innovation Fund of Khon Kaen University. RS’s visit to the NHMD,
Conceptualization: TW, RS. Data curation: RS, TW. Formal analysis: RS. Funding acquisition: RS. Investigation: RS. Methodology: TW, RS. Project administration: RS. Resources: NL, CS, RS. Supervision: CS, TW. Validation: CS, TW. Visualization: RS, NL. Writing - original draft: RS. Writing - review and editing: RS, TW, CS.
Ruttapon Srisonchai https://orcid.org/0000-0002-7142-0999
Natdanai Likhitrakarn https://orcid.org/0000-0002-1306-317X
Chirasak Sutcharit https://orcid.org/0000-0001-7670-9540
Thomas Wesener https://orcid.org/0000-0002-2028-3541
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Estimates of cytochrome c oxidase I (COI) sequence divergences (uncorrected p-distances) within and among Zephronia species and related genera (Sphaerobelum, Prionobelum and Cryxus)
Data type: xlsx
Phylogenetic tree based on maximum likelihood (ML) approach of genus Zephronia and related genera (Sphaerobelum, Prionobelum and Cryxus)
Data type: pdf